JP2020012817A - Analysis method and analysis system - Google Patents

Abstract

To provide an analysis method that excels in the accuracy of analyzing a sample.SOLUTION: Provided is an analysis method that uses a microchip which is provided with a capillary passage and a sample reservoir unit connected to the capillary passage, with the capillary passage filled with a first liquid for cataphoresis, and in which a second liquid containing the sample is retained in the sample reservoir unit, and includes: a pressurizing step for pressurizing the first liquid to the inside of the capillary passage from the side of the capillary passage opposite the side where it connects to the sample reservoir unit; and a separation step for applying a voltage to the inside of the sample reservoir unit where the second sample is retained and the capillary passage filled with the first liquid after the pressuring step, and when a sample component included in the second liquid is thereby moved inside of the capillary passage, separating the component inside of the capillary passage.SELECTED DRAWING: None

Description

  The present invention relates to an analysis method and an analysis system.

  2. Description of the Related Art In the field of clinical testing, sample analysis by capillary electrophoresis has been conventionally performed. In recent years, sample analysis by electrophoresis using a microchip has been performed for miniaturization and simplification of the apparatus. For example, the microchip includes a capillary tube, a holding tank for holding various liquids, and the like.

  For example, when analyzing blood proteins such as hemoglobin by electrophoresis using a microchip, irradiating the capillary tube with light, detecting light transmitted through the capillary tube, and analyzing a sample moving in the capillary tube. I have. In addition, from the viewpoint of increasing the analysis accuracy of the sample, the capillary tube is filled with the electrophoresis running solution, and the sample is introduced into the introduction tank connected to the capillary tube. It has been proposed to generate a shear flow at a connection with a tank to form an interface between a sample and an electrophoresis solution at the connection (for example, see Patent Document 1).

JP-A-2006-057289

  For example, when a capillary tube is filled with an electrophoresis liquid and a sample is introduced into an introduction tank connected to the capillary tube, a part of the sample introduced into the introduction tank moves into the capillary tube, and the sample and the electrophoresis solution are mixed with each other. May not be formed well. At this time, if the sample is analyzed by electrophoresis, the analysis accuracy of the sample may be deteriorated.

  An object of the present invention is to provide an analysis method and an analysis system that are excellent in sample analysis accuracy.

Specific means for achieving the above-mentioned object are as follows, for example.
<1> A capillary channel and a sample reservoir connected to the capillary channel are provided, a first liquid for electrophoresis is filled in the capillary channel, and a second liquid containing a sample is stored in the sample reservoir. A pressurizing step of pressurizing the first liquid into the capillary flow path from a side of the capillary flow path opposite to a side connected to the sample storage section, using the microchip stored in the section; After the step, by applying a voltage to the sample storage section in which the second liquid is stored and the capillary channel filled with the first liquid, the sample contained in the second liquid is applied. A separation step of moving the components therein in the capillary flow path and separating the components in the capillary flow path.
<2> Before the pressurizing step, a voltage was applied to the sample storage section and the capillary flow path, and the capillary flow path filled with the first liquid and the second liquid were stored. The method further includes a confirmation step of confirming that the sample reservoir is electrically connected, wherein in the confirmation step, a component in the sample contained in the second liquid moves in the capillary channel. The analysis method described in <>.
<3> a filling step of filling the capillary channel with the first liquid, and a supplying step of supplying the second liquid to the sample reservoir after the filling step and before the pressurizing step. The analysis method according to <1> or <2>, further comprising:
<4> In the pressurizing step, by pressurizing the first liquid into the capillary channel, the first liquid and the second liquid are connected to a connection portion between the capillary channel and the sample storage unit. The analysis method according to any one of <1> to <3>, which forms an interface with a liquid.
<5> The analysis method according to any one of <1> to <4>, further including a waiting step of waiting for a predetermined time after the pressurizing step and before the separating step.

<6> An arrangement section in which a microchip including a capillary channel and a sample storage section connected to the capillary channel is arranged, and an electrophoresis device in the capillary channel in the microchip arranged in the arrangement section. Filling means for filling the first liquid, supply means for supplying a second liquid containing a sample to the sample storage part in the microchip arranged in the disposing part, and the first means by the filling means The first liquid is supplied into the capillary channel from the side of the capillary channel opposite to the side connected to the sample storage portion, which is performed after the filling of the liquid and after the supply of the second liquid by the supply unit. And a controller that controls the pressurization of the sample, the sample storage unit storing the second liquid after pressurizing the first liquid in the capillary channel, and the second unit. By applying a voltage to the capillary channel filled with the liquid, the components in the sample contained in the second liquid move in the capillary channel, and the components in the capillary channel are An analysis system comprising: a separation unit configured to separate components.

  According to one embodiment of the present invention, it is possible to provide an analysis method and an analysis system that are excellent in sample analysis accuracy.

1 is a schematic configuration diagram of a microchip used in an analysis method and an analysis system according to one embodiment of the present invention, in which (a) is a top view and (b) is a cross-sectional view taken along line C′-C ′ of (a). FIGS. 3A and 3B are schematic structural views of a microchip used in the analysis system of one embodiment of the present invention, in which FIG. 3A is a top view, FIG. 3B is a cross-sectional view taken along line III-III of FIG. FIG. 4 is a sectional view taken along line IV-IV. 1 is a cross-sectional view illustrating a schematic configuration of an analysis system according to one embodiment of the present invention. 5 is a flowchart illustrating an analysis method according to one embodiment of the present invention. 9 is a flowchart illustrating a modification of the analysis method according to one embodiment of the present invention. It is a graph which shows the result of having performed electrophoresis using a microchip.

Hereinafter, an analysis method and an analysis system of one embodiment of the present invention will be described.
In this specification, a numerical range represented by using “to” means a range including numerical values described before and after “to” as a lower limit and an upper limit.

[Analysis method]
An analysis method according to one embodiment of the present invention includes a capillary channel, and a sample storage unit connected to the capillary channel, wherein a first liquid for electrophoresis is filled in the capillary channel, and the first channel includes a sample. A microchip in which the second liquid is stored in the sample storage section, and pressurizing the first liquid into the capillary flow path from the side of the capillary flow path opposite to the side connected to the sample storage section. A pressure step and, after the pressurizing step, applying a voltage to the sample storage section in which the second liquid is stored and the capillary flow path filled with the first liquid, whereby the second liquid is applied. A component in the sample contained in the liquid moves in the capillary channel and separates the component in the capillary channel.

  When analyzing components in a sample by electrophoresis using a microchip, from the point of realizing stable measurement operation, from the sample storage part to the side opposite to the side connected to the sample storage part in the capillary channel Can be confirmed by applying a voltage. However, when the conduction is confirmed by applying a voltage, electrophoresis is inevitably performed in the capillary channel, and then, during a predetermined time during which the voltage application is stopped, the electrophoresis is performed with the first liquid as the electrophoresis liquid. The phenomenon that the interface with the second liquid containing the sample is mixed and blurred occurs. Then, since the sample is analyzed by electrophoresis after a predetermined time has elapsed after the continuity check, the component to be analyzed is not appropriately separated, and it can be accurately detected separately from other components. In some cases, such problems as inability to calculate the component ratio with high accuracy may occur, and the analysis accuracy of the sample may deteriorate.

  Further, when the capillary channel is filled with the first liquid, and then the second liquid containing the sample is supplied to the sample storage unit connected to the capillary channel, the second liquid is supplied with a momentum to supply the second liquid. Due to the directionality of the liquid, a part of the second liquid stored in the sample storage part may move into the capillary channel, and the interface between the first liquid and the second liquid may not be formed well. There is. At this time, if the sample is analyzed by electrophoresis, the analysis accuracy of the sample may be deteriorated as described above.

  On the other hand, in the analysis method of the present aspect, the first liquid in the capillary channel is filled in the capillary channel, and the second liquid containing the sample is stored in the sample storage unit using the microchip. A pressurizing step of pressurizing the liquid is performed, and a separating step of separating components in the capillary channel is performed after the pressurizing step. Thus, the analysis method of the present embodiment is excellent in sample analysis accuracy. The reason for this is that, by pressurizing the first liquid in the capillary flow path, the second liquid moved into the capillary flow path is sent back to the sample storage side, and the connection between the capillary flow path and the sample storage section is made. It is considered that an interface between the first liquid and the second liquid including the sample can be formed in the portion, and the sample can be accurately analyzed by applying a voltage in a state where the interface is formed. Can be In addition, by employing the analysis method of this aspect, even when conduction is confirmed by applying a voltage as described above, the analysis accuracy of the sample is excellent, and a simple conduction confirmation method, and the analysis accuracy of the sample can be improved. Can be compatible.

  Note that, in the analysis method of the present disclosure, only the second liquid that has moved into the capillary flow path is sent back to the sample storage section without returning the first liquid in the capillary flow path to the sample storage section. May be difficult in quality control. In this case, the second liquid and the first liquid near the boundary are also sent back to the sample storage side at the same time. However, since the volume of the capillary channel is very small with respect to the volume of the sample storage, Then, a very small amount of the first liquid moves to the sample reservoir. A very small amount of the first liquid that has moved to the sample reservoir is diluted by a large amount of the second liquid that is present in the sample reservoir. Further, since the volume of the capillary channel is very small, and the flow of fluid from the capillary channel to the sample reservoir due to pressurization remains, the second liquid existing in the sample reservoir is removed from the capillary channel. The interface between the first liquid and the second liquid can be formed at the opening of the capillary flow path at the connection between the sample storage section and the capillary flow path without moving.

  The microchip used in the analysis method of this embodiment is used for analyzing a substance (preferably a biological substance) in a sample by capillary electrophoresis.

Samples used in the analysis method of the present embodiment include biological samples, environmental samples, metals, chemical substances, pharmaceuticals, and the like. The specimen derived from the living body is not particularly limited, and examples thereof include urine, blood, hair, saliva, sweat, and nails. Examples of the blood sample include red blood cells, whole blood, serum, plasma, and the like. Examples of the living body include humans, non-human animals, plants, and the like. Examples of the non-human animals include mammals other than humans, amphibian reptiles, seafood, insects, and the like. The sample derived from the environment is not particularly limited, and examples thereof include food, water, soil, air, and air. Examples of the food include fresh foods and processed foods. Examples of the water include drinking water, groundwater, river water, seawater, domestic wastewater, and the like. The sample is preferably blood collected from a human body or the like.
Further, as a component contained in the sample and to be analyzed by the analysis method of the present embodiment (hereinafter, also referred to as “component contained in the sample”), a biological substance is preferable. Hemoglobin is also preferable among the biological substances.
More specifically, the sample is blood, and the component contained in the sample is preferably a biological substance, the sample is blood, and the component contained in the sample is more preferably hemoglobin. .
Further, for example, a sample that is diluted by suspending, dispersing, or dissolving it in a diluent described below may be used as the second liquid.

  Components contained in blood and to be analyzed by the analysis method of the present embodiment include hemoglobin (Hb), albumin (Alb), globulin (α1, α2, β, γ globulin), fibrinogen and the like. Examples of hemoglobin include normal hemoglobin (HbA0), glycated hemoglobin, modified hemoglobin, fetal hemoglobin (HbF), and mutant hemoglobin. Examples of the glycated hemoglobin include hemoglobin A1a (HbA1a), hemoglobin A1b (HbA1b), hemoglobin A1c (HbA1c), hemoglobin A1d1 (HbA1d1), hemoglobin A1d2 (HbA1d2), and hemoglobin H1AbH1AbH1Ab. And the like. Examples of hemoglobin A1c include stable HbA1c (S-HbA1c) and unstable HbA1c (L-HbA1c). Examples of the modified hemoglobin include carbamylated Hb and acetylated Hb. Examples of the mutant hemoglobin include hemoglobin C (HbC), hemoglobin D (HbD), hemoglobin E (HbE), and hemoglobin S (HbS).

  A microchip used in the analysis method of this embodiment will be described with reference to FIG. FIGS. 1A and 1B are schematic structural views of a microchip used in the analysis method of one embodiment of the present invention. FIG. 1A is a top view, and FIG. 1B is a cross-sectional view taken along line C′-C ′ of FIG. As shown in FIG. 1, the microchip 100 includes a sample storage unit 1, an electrophoresis solution storage unit 3, a capillary channel 2, and a detection unit 4. The microchip 100 may be manufactured, for example, by joining a pair of base members, which are plate members having a substantially rectangular shape. More specifically, a base material having a through-hole corresponding to the sample storage part 1 and the electrophoresis liquid storage part 3 and a base material having a fine groove corresponding to the capillary flow path 2 are formed such that both ends of the groove are 2 The microchip 100 may be manufactured by bonding so as to face a part of the two through holes.

  Examples of the material of the base material constituting the microchip include glass, fused silica, and resin. From the viewpoint of cost, ease of processing, and ease of immobilization of the cationic polymer, resin is preferable. . Examples of the resin include acrylic resins such as polymethyl methacrylate (PMMA), polymethyl methacrylate, polycarbonate, polyvinylidene chloride, cyclic polyolefin, polyetheretherketone, polystyrene, polytetrafluoroethylene (PTFE), cycloolefin, polypropylene, and Polyethylene and the like are exemplified, and polymethyl methacrylate is preferred from the viewpoint of excellent light transmittance.

  The microchip used in the analysis method of the present embodiment may be a disposable type analysis chip that is not reused.

  The sample storage section 1 is a tank to which a second liquid containing a component such as a biological substance is supplied from an opening, and stores the supplied second liquid. The sample storage section 1 is connected to an end of the capillary flow path 2, and more specifically, is connected to an end of the capillary flow path 2 opposite to the electrophoresis liquid storage section 3 side.

  The second liquid may be a liquid containing the above-described sample, and may be the sample itself, or may be a liquid obtained by diluting the sample with a diluent.

  The main component of the diluting liquid is not particularly limited, and includes water and physiological saline. As a preferable example, a substance that can be contained in a first liquid for electrophoresis described later may be added to the diluting liquid. Further, the diluting liquid may be, for example, a liquid obtained by adding a cathodic group-containing compound to a main agent. Examples of the cathodic group-containing compound include cathodic group-containing polysaccharides, and more specifically, sulfated polysaccharides, carboxylated polysaccharides, sulfonated polysaccharides, and phosphorylated polysaccharides. As the carboxylated polysaccharide, alginic acid and a salt thereof (for example, sodium alginate) are preferable. As the sulfated polysaccharide, for example, chondroitin sulfate is preferable. There are seven types of chondroitin sulfate, A, B, C, D, E, H, and K, and any of them may be used. The concentration of the anionic group-containing compound (chondroitin sulfate) is preferably, for example, in the range of 0.01% by mass to 5% by mass.

  The electrophoresis liquid storage unit 3 is a tank to which the first liquid for electrophoresis is supplied from the opening and stores the supplied first liquid. The electrophoresis liquid reservoir 3 is connected to an end of the capillary channel 2, and more specifically, to an end of the capillary channel 2 on the side opposite to the sample reservoir 1 side. Further, the first liquid stored in the electrophoresis liquid storage unit 3 is configured to be filled into the capillary channel 2 by pressurization.

  The first liquid, which is an electrophoresis liquid used for electrophoresis, is supplied from an opening to the electrophoresis liquid storage unit 3 and is a medium that generates an electroosmotic flow in electrophoresis by being filled in the capillary channel 2. . The first liquid includes water, saline, and the like. Preferably, the first liquid comprises an acid. Examples of the acid include citric acid, maleic acid, tartaric acid, succinic acid, fumaric acid, phthalic acid, malonic acid, and malic acid. Further, the first liquid preferably contains a weak base. Examples of the weak base include arginine, lysine, histidine, tris and the like. The pH of the first liquid is preferably, for example, in the range of pH 4.5 to pH6. Examples of the first liquid buffer include MES, ADA, ACES, BES, MOPS, TES, HEPES, and the like. Further, the cathodic group-containing compound described in the description of the diluent may be added to the first liquid. The concentration of the anionic group-containing compound (such as chondroitin sulfate) is preferably, for example, in the range of 0.01% by mass to 5% by mass.

  The capillary flow path 2 connects the sample storage unit 1 and the electrophoretic liquid storage unit 3 and is a flow path for analyzing a component in the second liquid supplied to the sample storage unit 1, and more specifically. Is a capillary tube for performing analysis of an analysis target by electrophoresis.

  The size of the capillary channel 2 is not particularly limited. For example, it is preferable that the depth is 25 μm to 100 μm, the width is 25 μm to 100 μm, and the length is 5 mm to 150 mm.

  The capillary channel 2 may be provided with a charge, for example, a positive charge, on the wall surface in order to enhance the analysis performance. The method for imparting a positive charge is not particularly limited, and a cationic polymer is immobilized on a region corresponding to the capillary channel 2 in the pair of base materials before the pair of base materials is joined. Just do it.

  The detection unit 4 is used for inputting and outputting light to be analyzed when performing electrophoretic analysis on the microchip 100, and is used for, for example, absorbance measurement. The detection unit 4 is formed at a position facing at least a part of the capillary channel 2. The position of the detection unit 4 may be appropriately determined according to the length of the capillary channel 2 and the like.

  Hereinafter, each step of the analysis method of this embodiment using the microchip 100 will be described with reference to FIG. Note that the analysis method of the present invention is not limited to the method including all the following steps.

(Filling process)
The analysis method of the present embodiment includes a filling step (S1) of filling the capillary channel 2 with the first liquid using the microchip 100 described above. In the filling step (S1), for example, the first liquid is supplied to the electrophoresis liquid storage section 3, and the first liquid stored in the electrophoresis liquid storage section 3 is pressurized to cause the first liquid to flow into the capillary channel 2. May be supplied and filled.

(Supply process)
The analysis method of this aspect includes a supply step (S2) of supplying the second liquid to the sample storage unit 1 after the filling step (S1). In the analysis method of the present invention, a filling step of filling the capillary channel with the first liquid may be performed after the supplying step of supplying the second liquid to the sample storage unit.

(Confirmation process)
In the analysis method according to this aspect, after the supply step (S2), a voltage is applied to the sample storage unit 1 and the capillary channel 2 so that the capillary channel 2 filled with the first liquid and the second liquid are stored. A confirmation step (S3) for confirming that the conducted sample storage unit 1 is electrically connected is included. In the confirmation step (S3), the components in the sample contained in the second liquid move into the capillary channel 2.

  For example, in the confirmation step (S3), the anode is brought into contact with the second liquid stored in the sample storage unit 1, and the cathode is brought into contact with the first liquid stored in the electrophoresis liquid storage unit 3 to apply a voltage. You may. The voltage to be applied is not particularly limited, and may be, for example, 0.5 kV to 20 kV, 0.5 kV to 10 kV, or 0.5 kV to 5 kV. The confirmation step (S3) may be ended when it is confirmed that a predetermined current (for example, 5 μA or more) has flowed when a voltage within the above-described numerical range is applied.

  In the confirmation step (S3), the cathode is brought into contact with the second liquid stored in the sample storage unit 1, and the anode is brought into contact with the first liquid stored in the electrophoresis liquid storage unit 3 to apply a voltage. Alternatively, the components in the sample contained in the second liquid may move into the capillary channel 2. In this case, it is preferable to use an alkaline solution containing a cationic polymer as the first liquid and the second liquid. As the alkaline solution containing a cationic polymer, for example, an alkaline solution containing a cationic polymer described in Japanese Patent No. 6052927 can be used.

  After the confirmation step (S3), as described later, at least one of the discharge and the suction using the pump is performed once, or the discharge and the suction using the pump are repeated to store the sample in the sample storage unit. The second liquid may flow.

(Pressure process)
In the analysis method of the present embodiment, after the confirmation step (S3), the pressurizing step of pressurizing the first liquid in the capillary flow path 2 from the side of the capillary flow path 2 opposite to the side connected to the sample storage unit 1 ( S4). For example, in the pressurizing step (S4), the first liquid in the capillary channel 2 may be pressurized from the side of the capillary channel 2 opposite to the side connected to the sample storage unit 1, and more specifically, Alternatively, the first liquid stored in the electrophoresis liquid storage unit 3 may be pressurized to resupply the first liquid into the capillary channel 2. The condition for pressurizing the first liquid stored in the electrophoresis liquid storage unit 3 may be appropriately adjusted depending on the voltage application condition in the above-described confirmation step, the size of the capillary channel 2, and the like, for example, at 1 kPa to 20 kPa. Pressurization may be performed for 0.1 second to 5 seconds. As a pressurization method, a method is used in which the first liquid is pressurized by air pressure and sent into the capillary flow path 2, and the second liquid moved into the capillary flow path 2 is sent back to the sample storage unit 1 side. Is also good.

(Separation process)
Next, in the analysis method of this aspect, after the pressurizing step (S4), a voltage is applied to the sample storage unit 1 in which the second liquid is stored and the capillary channel 2 filled with the first liquid. Accordingly, a separation step (S5) of moving the components in the sample contained in the second liquid in the capillary channel 2 and separating the components in the capillary channel 2 is included.

  For example, in the separation step (S5), the anode is brought into contact with the second liquid stored in the sample storage unit 1, and the cathode is brought into contact with the first liquid stored in the electrophoretic liquid storage unit 3 to apply a voltage. You may. As a result, an electroosmotic flow is generated in the capillary channel 2, the second liquid is introduced from the sample reservoir 1 into the capillary channel 2, and the second liquid flows from the sample reservoir 1 to the electrophoresis liquid reservoir 3. When moving, the components in the second liquid are separated. The voltage to be applied is, for example, preferably from 0.5 kV to 20 kV, more preferably from 0.5 kV to 10 kV, and still more preferably from 0.5 kV to 5 kV. As described above, even when a voltage is applied by bringing the cathode into contact with the second liquid stored in the sample storage unit 1 and bringing the anode into contact with the first liquid stored in the electrophoresis liquid storage unit 3, Often, in this case, it is preferable to use an alkaline solution containing a cationic polymer as the first liquid and the second liquid.

(Detection process)
The analysis method of this aspect may include a detection step of detecting a component separated in the capillary channel. For example, the separated components may be detected by an optical method such as absorbance measurement, or may be detected by irradiating light from the detection unit 4 and measuring the absorbance. Alternatively, the amount of change in absorbance per unit time may be determined from the measured absorbance.

  When the component in the sample contained in the second liquid is hemoglobin, for example, it is preferable to measure the absorbance at a wavelength of 415 nm. Also, the peak ratio, the peak area, and the like of the electropherogram obtained by measuring the absorbance may be calculated to determine the component ratio and the like in the second liquid.

[Modification]
A modification of the analysis method according to the present embodiment will be described. The analysis method of the modified example is different from the above-described analysis method in that the analysis method includes a standby step of waiting for a predetermined time after the pressurizing step and before the separation step. Hereinafter, only differences from the above-described analysis method of the present embodiment will be described.

  In the pressurizing step (S4), based on the voltage application conditions in the confirmation step (S3), the size of the capillary channel 2, etc., only the second liquid that has theoretically been moved into the capillary channel 2 is stored in the sample. It is desirable to send it back to unit 1. However, it is difficult to accurately return only the second liquid that has moved into the capillary channel 2 to the sample storage unit 1 due to conditions such as a difference in the individual microchips 100 and an environmental temperature. Therefore, in the pressurizing step (S4), it is preferable that the second liquid and the first liquid near the boundary are also simultaneously sent back to the sample storage unit 1 side. Since the volume of the capillary channel 2 is very small with respect to the volume of the sample storage unit 1, a very small amount of the first liquid moves to the sample storage unit 1. A very small amount of the first liquid that has moved to the sample storage unit 1 is diluted by a large amount of the second liquid that exists in the sample storage unit 1. Furthermore, since the volume of the capillary channel 2 is very small, and the flow of the fluid from the capillary channel 2 to the sample reservoir 1 due to pressurization remains, the second liquid existing in the sample reservoir 1 Does not move to the capillary channel 2, and an interface between the first liquid and the second liquid is formed at the opening of the capillary channel 2 at the connection between the sample storage unit 1 and the capillary channel 2. be able to.

  In the pressurizing step (S4), when the second liquid and the first liquid near the boundary are also simultaneously sent back to the sample storage unit 1 side, the first liquid near the boundary is returned to the sample storage unit 1 side. The extent to which the sheet is fed also varies depending on the standby time in a standby step (S5) described later. For example, when a relatively large amount of the first liquid near the boundary is sent back to the sample storage unit 1 side, it is preferable to secure a longer standby time.

(Standby process)
The analysis method of this aspect includes a waiting step (S5) of waiting for a predetermined time after the pressurizing step (S4) and before the separation step (S6) described below. The waiting time is preferably, for example, 0.1 second to 10 seconds, and more preferably 0.2 second to 3 seconds. Thereby, even if the first liquid moves to the sample storage unit 1 side, the first liquid is diluted by a large amount of the second liquid existing in the sample storage unit 1, and the interface between the first liquid and the second liquid is reduced. At the connection between the sample reservoir 1 and the capillary channel 2, it can be formed at the opening of the capillary channel. Note that a phenomenon may occur in which the second liquid flows from the sample storage unit 1 into the capillary channel 2 again. Therefore, the standby time is preferably shorter than the time when this phenomenon occurs.

  The volume of the first liquid sent back to the sample storage unit 1 in the pressurizing step (S4) is diluted into the second liquid in the sample storage unit 1 after the elapse of the standby time in the standby step (S5). It is preferable that the amount is in the order of magnitude. For example, the liquid (the first liquid and the second liquid) that is sent back to the sample storage unit 1 in the pressurizing step (S4) with respect to the amount of the second liquid supplied to the sample storage unit 1 in the supply step (S2) Of the total amount of the liquid (the amount of the second liquid supplied to the sample storage unit 1) may be 0.0001 to 0.1. Alternatively, the volume ratio of the total amount of liquid sent back to the sample storage unit 1 in the pressurizing step (S4) with respect to the volume of the capillary channel 2 (total amount of liquid sent back / volume of capillary channel 2) May be 0.1 to 100.

(Separation process)
Next, the analysis method of the modified example includes a separation step (S6) after the standby step (S5). The separation step (S6) is the same as the above-described separation step (S5), and a description thereof will not be repeated.

〔kit〕
The microchip used in this embodiment may be combined with a cartridge for storing a liquid for analysis to form a kit. The kit may include, for example, a microchip used in this embodiment, and a cartridge for storing the diluent and the first liquid, respectively.

[Analysis system]
Further, the analysis system according to the present aspect further includes an arrangement section in which a microchip including a capillary channel and a sample storage section connected to the capillary channel is arranged, and the capillary flow in the microchip arranged in the arrangement section. Filling means for filling a first liquid for electrophoresis in a passage, supply means for supplying a second liquid containing a sample to the sample storage part in the microchip arranged in the arrangement part, After the filling of the first liquid by the means and after the supply of the second liquid by the supply means, the capillary flow path enters the capillary flow path from the side opposite to the side connected to the sample storage section of the capillary flow path. And a controller for controlling the pressurization of the first liquid, and before the second liquid is stored after the first liquid is pressurized in the capillary channel. By applying a voltage to the sample reservoir and the capillary channel filled with the first liquid, the components in the sample contained in the second liquid move in the capillary channel, Separating means for separating the components in the capillary channel. The analysis system of this embodiment is an embodiment of an analysis system used for performing the above-described analysis method of this embodiment, and is excellent in sample analysis accuracy.

  The analysis system of this aspect includes an arrangement unit to which the above-described microchip is attached. The microchip attached to the placement unit includes, for example, a sample storage unit, a capillary channel connected to the sample storage unit, and the like. The microchip may include an electrophoresis liquid reservoir connected to the capillary channel on the side opposite to the sample reservoir. The first liquid is filled into the capillary channel by the filling means. When a microchip having an electrophoresis liquid reservoir is used, the first liquid for electrophoresis is supplied to the electrophoresis liquid reservoir by the filling means, and the first liquid is supplied into the capillary channel by pressurization and filled. Is done. Further, the second liquid is supplied to the sample storage unit by the supply unit, and the second liquid is stored in the sample storage unit. The filling means and the supply means may be, for example, pumps described later.

  According to the analysis system of this aspect, in the microchip in which the first liquid is filled in the capillary flow path and the second liquid is stored in the sample storage section, the first liquid is introduced into the capillary flow path by the control section. To control the pressurization. Thus, similarly to the above-described analysis method, an interface between the first liquid and the second liquid can be formed, and a sample can be accurately analyzed by applying a voltage in a state where the interface is formed. . In addition, as a method of pressurizing the first liquid into the capillary channel, the first liquid is pressurized by air pressure and sent into the capillary channel, and the second liquid moved into the capillary channel is compressed. A method of feeding the sample backward to the sample storage unit 1 may be used. Also, the filling means may have both a configuration in which the first liquid is pressurized and supplied into the capillary flow path to be filled, and a configuration in which the first liquid is pressurized and re-supplied into the capillary flow path. Good. Alternatively, a configuration in which the first liquid is pressurized by a pressurizing unit different from the filling unit and resupplied into the capillary channel may be employed.

  Further, in the analysis system according to this aspect, after the first liquid is pressurized in the capillary channel by the separation unit, the sample storage unit storing the second liquid and the first liquid are filled. By applying a voltage to the inside of the capillary channel, components in the sample contained in the second liquid move in the capillary channel and separate components in the capillary channel. The separation means applies a predetermined voltage in the capillary channel, and applies, for example, an anode inserted into the sample storage section, a cathode inserted into the electrophoresis running liquid storage section, and a voltage to the anode and the cathode. A voltage application unit is included.

  Further, the analysis system according to the present aspect may be configured such that after the first liquid is filled by the filling means and after the supply of the second liquid by the supply means, the first liquid in the capillary flow path is pressurized. A confirmation means may be provided for confirming that the capillary flow path filled with is connected to the sample storage section in which the second liquid is stored. The above-described voltage applying unit may also serve as the checking unit.

  The analysis system according to this aspect may include a detection unit that detects the separated component. The detection means may be, for example, a means for performing the above-mentioned separated components by an optical method such as absorbance measurement. The detecting unit may be, for example, a light emitting unit and a measuring unit.

  The light emitting unit is, for example, a part that emits light for measuring absorbance and irradiates the light to the detection unit of the microchip. The light emitting unit includes, for example, an LED chip, an optical filter, and a lens that emit light in a predetermined wavelength range. Further, the light emitting section may include a slit.

  The measurement unit is, for example, a unit that receives light emitted to the detection unit of the microchip and measures the absorbance. The measurement unit includes, for example, a photodiode, a photo IC, and the like.

  Further, the analysis system of the present embodiment may include a diluent tank, an electrophoresis tank, a pump, and the like. Further, the control unit may control a separating unit such as a filling unit, a supplying unit, and a voltage applying unit, and a detecting unit such as a light emitting unit and a measuring unit. Further, the control unit controls the filling means or the pressurizing means to pressurize the first liquid and resupply the first liquid into the capillary channel, and then waits for the above-mentioned predetermined time before controlling the separating means to control the second liquid. The components in the sample contained in the liquid may be moved into the capillary channel and the components may be separated in the capillary channel.

  The diluent tank is, for example, a tank that stores a diluent for diluting a sample. For example, after the diluent supplied from the diluent tank and the sample are mixed in the mixing tank, a liquid (second liquid) obtained by diluting the sample may be supplied to the sample storage unit. In this case, the microchip used in the analysis system of the present embodiment may include a mixing tank for mixing the diluent supplied from the diluent tank with the sample containing the analysis target.

  The electrophoresis liquid tank is, for example, a tank that stores the first liquid for electrophoresis supplied to the electrophoresis liquid storage unit.

  The pump supplies, for example, a diluting liquid to the mixing tank by applying pressure, or supplies a first liquid to the electrophoretic liquid storage unit by applying pressure, and fills the capillary liquid with the first liquid. It is a part for. The pump may supply a liquid (second liquid) obtained by diluting the sample containing the analysis target in the mixing tank to the sample storage unit. In addition, the second liquid stored in the sample storage unit may be caused to flow by performing at least one of the discharge and the suction using the pump once, or repeating the discharge and the suction using the pump. After confirming that the capillary channel filled with the first liquid is electrically connected to the sample storage unit storing the second liquid by a checking unit such as a voltage applying unit, the second liquid May be fluidized.

  FIG. 2 shows a microchip having a mixing tank and having a configuration capable of flowing the second liquid stored in the sample storage section. The microchip 200 shown in FIG. 2 includes a mixing tank 5 for mixing a diluent and a sample containing an analysis target, and a sample storage unit 11 having two openings and capable of flowing a second liquid. Prepare. When the second liquid stored in the sample storage unit 11 is caused to flow, for example, the second liquid stored in the sample storage unit 11 may be subjected to at least one of discharge and suction using a pump once. Alternatively, reciprocation in the y direction in FIG. 2 may be performed by repeating discharge and suction using a pump.

  When the second liquid stored in the sample storage unit 11 is caused to flow by repeating discharge and suction using a pump, the pump is strongly idled, and then the pump and the sample storage unit 11 of the microchip 200 are connected. Then, it is preferable that the second liquid stored in the sample storage unit 11 is caused to flow. Thereby, the pressure at the time of operating the pump is easily stabilized.

  The control unit only needs to control at least the pressurization of the first liquid into the capillary channel after the filling of the first liquid by the filling unit and after the supply of the second liquid by the supply unit. The control unit may control each of the above-described components in the analysis system. For example, the control unit may control each of the components in the analysis system such that each of the components performs the steps illustrated in FIG. 4 in this order. May be used. The control unit includes, for example, a CPU, a memory, an interface, and the like.

  The analysis system of this embodiment will be described with reference to FIG. FIG. 3 is a cross-sectional view illustrating a schematic configuration of the analysis system of one embodiment of the present invention. The analysis system 300 shown in FIG. 3 includes a placement unit 12 to which a microchip 200 is attached, an anode 6, a cathode 7, a pump 8, a control unit 10, a photodiode 13, an LED chip 14, and an optical filter 15. And a lens 16 and a slit 17.

  The anode 6 is inserted into the sample storage unit 11, and the cathode 7 is inserted into the electrophoresis solution storage unit 3. In addition, the LED chip 14 irradiates light to the detection unit 4 of the microchip 200, and the photodiode 13 receives light irradiated to the detection unit 4 of the microchip 200, and measures the absorbance.

  The pump 8 supplies the first liquid to the electrophoresis liquid storage unit 3, and then supplies and fills the first liquid into the capillary channel 2 by pressurization, and also applies a voltage to the capillary channel 2 by applying a voltage. Before the components are separated, the first liquid is resupplied into the capillary channel 2 by pressurization.

  The control unit 10 controls each component of the analysis system 300. For example, the control unit 10 controls the filling and resupply of the first liquid by the filling unit 8, the control of the voltage applied to the anode 6 and the cathode 7, the LED, and the like. Control of light emitted from the chip 14, measurement of absorbance based on light received by the photodiode 13, and the like may be performed. Further, the control unit 10 may perform control of discharge and suction of a pump, supply of a diluent, a second liquid, and the like, control of flow, and the like. Further, the control unit 10 may be configured to control pressurization of the first liquid by a pressurizing unit different from the filling unit 8.

  As described above, the analysis method and the analysis system according to one embodiment of the present invention have been described. However, the present invention is not limited to these embodiments, and various modifications may be made based on the knowledge of those skilled in the art without departing from the spirit of the present invention. Improvements, changes, and modifications may be made. In addition, the items described in the items of the analysis method and the analysis system according to one embodiment of the present invention may be appropriately combined.

  Next, one embodiment of the present invention will be described based on the following examples, but the present invention is not limited to the following examples.

[Example 1]
<Preparation of microchip>
In this example, a microchip having each configuration satisfying the following conditions was prepared. The inner wall of the capillary channel is coated with polydiallyldimethylammonium chloride (PDADMAC: manufactured by Sigma).
Sample storage part: volume 20μL
Electrophoresis liquid reservoir: volume 10 μL
Capillary flow channel: depth 0.04 mm x width 0.04 mm x length 30 mm (Optical detection is performed at the detection unit 20 mm from the sample storage unit side)

<Preparation of first liquid and second liquid>
As the first liquid, 40 mM citric acid, 1.25% (w / v) chondroitin sulfate C-sodium, 0.1% (w / v) LS-110 (manufactured by Kao Corporation), 0.1% (w / V) A solution prepared using sodium azide and adjusted to pH 5.0 with dimethylaminoethanol (for pH adjustment) was used.
As a sample, 95 μL of whole blood collected from a human body was used.
As a diluent, 40 mM citric acid, 1.0% (w / v) chondroitin sulfate C-sodium, 500 mM NDSB-201 (manufactured by Anatarase), 0.1% (w / v) LS-110 (manufactured by Kao Corporation) ) And 0.1% (w / v) sodium azide and adjusted to pH 6.0 with dimethylaminoethanol (for pH adjustment).
The sample was diluted 41 times with the diluent to prepare a second liquid.

<Capillary electrophoresis>
Hemoglobin in the sample was analyzed according to the following procedure.
1) The microchip was set on a special device manufactured by ARKRAY.
2) The first liquid was added to the electrophoresis liquid reservoir of the microchip, and the first liquid was supplied and filled into the capillary channel by pressurization.
3) The second liquid was added to the sample storage.
4) The anode was brought into contact with the sample storage part and the cathode was brought into contact with the electrophoresis running liquid storage part, a voltage of about 3000 V was applied, and a current of 5 μA or more was confirmed to flow.
5) The second liquid stored in the sample storage section was caused to flow by discharge using a pump.
6) The first liquid stored in the electrophoresis liquid storage unit was pressurized under the conditions of 6 kPa and 1 second, and the first liquid was supplied again into the capillary channel.
7) The anode was brought into contact with the sample reservoir and the cathode was brought into contact with the electrophoresis running fluid reservoir, and electrophoresis was started at a voltage of about 1500 V and a constant current control of 70 μA.
8) The absorbance at 415 nm was measured by the detection unit to obtain an electropherogram. Electrophoresis was performed for 35 seconds.

[Example 2]
The first liquid stored in the electrophoresis liquid storage unit is pressurized under the conditions of 10 kPa and 1 second in the above-mentioned “<6) of capillary electrophoresis”, and the first liquid is injected into the capillary channel. Electrophoresis was performed in the same manner as in Example 1 except that the supply was performed again.

[Comparative Example 1]
Electrophoresis was performed in the same manner as in Example 1, except that the above-mentioned “<6) of Capillary Electrophoresis” was not performed.

  The results of the capillary electrophoresis in Examples 1 and 2 and Comparative Example 1 are as shown in FIG. In FIG. 6, the horizontal axis represents the detection time (second), and the vertical axis represents the amount of change in absorbance (mAbs / second). As shown in FIGS. 6A and 6B, in the electropherograms of Example 1 and Example 2, the peaks of the analytical components S-HbA1c and HbA1d1 are separated from each other by a time interval. And clearly appeared. On the other hand, as shown in FIG. 6C, in the electropherogram of Comparative Example 1, the peaks of S-HbA1c and HbA1d1, which are the analysis components, did not appear individually and clearly. Therefore, the analysis methods of Example 1 and Example 2 were superior to the analysis method of Comparative Example 1 in the analysis accuracy of the sample.

  1, 11 sample storage section, 2 capillary flow path, 3 electrophoresis liquid storage section, 4 detection section, 5 mixing tank, 6 anode, 7 cathode, 8 pump (filling means), 10 control section, 12 placement section, 13 photodiode (Measurement unit), 14 LED chip (light-emitting unit), 15 optical filter, 16 lens, 17 slit, 100, 200 microchip, 300 analysis system

Claims (6)

A capillary channel, and a sample reservoir connected to the capillary channel, wherein a first liquid for electrophoresis is filled in the capillary channel, and a second liquid containing a sample is stored in the sample reservoir. Using the microchip
A pressurizing step of pressurizing the first liquid into the capillary flow path from a side of the capillary flow path opposite to a side connected to the sample storage unit;
After the pressurizing step, by applying a voltage to the sample storage section in which the second liquid is stored and the capillary channel filled with the first liquid, the sample liquid is contained in the second liquid. A component in the sample to be moved in the capillary channel, a separation step of separating the component in the capillary channel,
Analysis methods including: Before the pressurizing step, a voltage is applied to the sample storage section and the capillary flow path, and the capillary flow path filled with the first liquid and the sample storage in which the second liquid is stored Further including a confirmation step of confirming that the part is electrically connected,
2. The analysis method according to claim 1, wherein in the confirmation step, components in the sample included in the second liquid move in the capillary channel. 3. A filling step of filling the first liquid into the capillary channel;
After the filling step and before the pressurizing step, a supplying step of supplying the second liquid to the sample storage unit,
The analysis method according to claim 1 or 2, further comprising:   In the pressurizing step, by pressurizing the first liquid into the capillary flow path, a connection between the first liquid and the second liquid is formed at a connection between the capillary flow path and the sample storage unit. The analysis method according to any one of claims 1 to 3, wherein an interface is formed.   The analysis method according to any one of claims 1 to 4, further comprising a waiting step of waiting for a predetermined time after the pressurizing step and before the separating step. A capillary channel, and an arrangement unit where a microchip including a sample storage unit connected to the capillary channel is arranged,
Filling means for filling a first liquid for electrophoresis in the capillary flow channel in the microchip arranged in the disposing portion;
Supply means for supplying a second liquid containing a sample to the sample storage section in the microchip arranged in the arrangement section,
The capillary flow path, which is performed after the filling of the first liquid by the filling means and after the supply of the second liquid by the supply means, from a side of the capillary flow path opposite to a side connected to the sample storage section. A control unit for controlling the pressurization of the first liquid into the inside,
After pressurizing the first liquid in the capillary flow path, applying a voltage to the sample storage section in which the second liquid is stored and to the capillary flow path filled with the first liquid. By this means, separation means for moving components in the sample contained in the second liquid in the capillary flow path and separating the components in the capillary flow path,
An analysis system comprising: